Immunogenic Composition Comprising Peptides Derived From Cytomegalovirus And The Use Thereof

Abstract

The present invention provides (poly)peptides, which are recognized by human cytomegalovirus (CMV)-specific immune cells. The present invention further provides a combination of multiple CMV (poly)peptides, comprising at least two different groups of (poly)peptides according to the invention as well as conjugates, comprising said (poly)peptides and/or immune adjuvants thereof. Furthermore, this invention provides mixtures, comprising said (poly)peptides and/or immune cells thereof, which are used to generate CMV-specific immune effector cells with high sensitivity and specificity. In addition, the present invention provides a preparation method of CMV-specific immune effector cells, by using said (poly)peptides, adjuvants, immune cells and/or mixtures thereof to generate anti-CMV immune response.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to an anti-herpes virus vaccine, particularly for producing the antigen composition of anti-herpes virus of immune effector cells. The present invention also relates to the use of the above antigen composition and the method for activating immune cells.

[0003] 2. Description of the Prior Arts

[0004] Herpes virus belongs to Herpesviridae. As currently known, eight kinds of viruses cause diseases; these viruses are called human herpes viruses (HHV), including: (1) human herpes cirus-1 (HHV-1), also known as herpes simplex virus-1 (HSV-1); (2) human herpes virus-2 (HHV-2), also known as herpes simplex virus-2 (HSV-2); (3) human herpes virus-3 (HHV-3), also known as Varicella Zoster Virus (VZV), which causes chicken pox and herpes zoster and is also called varicella virus; (4) human herpes virus-4 (HHV-4), also known as Epstein-Barr Virus (EBV), which is a lymphocytic virus, causes Burkitt's lymphoma and nasopharyngeal carcinoma; (5) human herpes virus-5 (HHV-5), also known as cytomegalovirus (CMV); (6) human herpes virus-6 (HHV-6), also known as roseolovirus, which causes the sixth syndrone including roseola infantum and exanthema subitum; (7) human herpes virus-7 (HHV-7), related to HHV-6 and causing similar symptoms; and (8) human herpes virus-8 (HHV-8), which is Genus Rhadinovirus, referred to as kSHV, and could be found in Kaposi's sarcoma.

[0005] Current management of herpes virus infections in transplant recipients includes prophylaxis or preemptive treatment with antiviral agents Taking Cytomegalovirus (CMV) as an example, for the former is positive in culture test or expresses virus active replication in test, to heal beforehand without any symptoms. The latter is aimed at serum positive for healing beforehand without any symptoms. There are several antiviral drugs available against CMV replication, including ganciclovir, foscarnet, and cidofovir. (Gandhi M K, et al., Lancet Infect. Dis., 2004, 4:725-38) Significant progress has been made in the control of CMV infection in transplant recipients with these agents.

[0006] However, these pharmacological strategies have limitations, e.g., drug toxicities, development of resistance, poor oral bioavailability, and low potency. Mortality remains high even though patients are treated with antiviral agents, especially when therapy is not initiated early in the course. (Gandhi M K, et al., Lancet Infect. Dis., 2004, 4:725-38; Ison M G, et al., Clin. Chest Med., 2005, 26:691-705; Gandhi M K, et al., Blood Rev., 2003, 17:259-64) In addition, resistance to antiviral agents emerges in patients after prolonged exposure to this agent (Ison M G, et al., supra; Biron K K., Antiviral Res., 2006, 71: 154-63).

[0007] Existing immune-therapies for herpes virus infected diseases use herpes virus antigen sources based on live viruses, herpes virus-infected cells, herpes virus gene expression vectors, or synthetic herpes virus proteins or peptides to prime antigen-presenting cells for the activation of herpes virus-specific immune response. The use of viruses or virus-infected cells as antigen source has potential risk of virus contamination and infection. The use of full-length herpes virus genes or proteins as antigen source has potential risk of induction of immune suppression or tolerance.

SUMMARY OF THE INVENTION

[0008] Given that the aforesaid drawbacks of the prior art such as virus-infection and immune tolerance, the objective of the present invention provides an immune therapy for herpes virus, which does not produce problems as which are caused by using live viruses, herpes virus-infected cells or full-length herpes virus proteins.

[0009] Therefore, in one aspect, the present invention provides an immunogenic composition containing peptides derived from CMV, which comprises: (a) at least one peptide fragment selected from CMV pp65-derived and CMV IE-1-derived peptide fragments; and (b) one or more peptide fragment(s) selected from the group consisting of CMV VGLB, CMV VPAP and CMVp100 polypeptide-derived peptide fragments.

[0010] In a second aspect, the present invention provides an immunogenic composition containing peptides derived from CMV, which comprises: (a) at least one peptide pool selected from CMV pp65 and CMV IE-1 polypeptides-derived peptide pools; and (b) one or more peptide pool(s) selected from the group consisting of CMV VGLB, CMV VPAP and CMVp100 polypeptide-derived peptide pool.

[0011] In a preferred embodiment of the immunogenic composition of the present invention, CMV pp65 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 1; CMV IE-1 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 2; CMV VGLB has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 3; CMV VPAP has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 4; and CMV p100 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 5.

[0012] Preferably, said immunogenic composition comprises CMV pp65, CMV IE-1, CMV VGLB, CMV VPAP and CMV p100 polypeptide-derived peptide pools.

[0013] In a third aspect, the present invention provides use of the immunogenic composition as in any of the aforesaid first to second aspects for ex vivo inducing immune response for herpes virus.

[0014] In a fourth aspect, the present invention provides use of the immunogenic composition as in any of the aforesaid first to second aspects for manufacturing medicament for treating herpes virus infection diseases.

[0015] In a fifth aspect, the present invention provides a method for ex vivo activating immune cell, comprising steps of: mixing an immune cell and an immunogenic composition, as in any of the aforesaid first to second aspects, to form a mixed culture; then incubating the mixed culture in a suitable medium to obtain the activated immune cell.

[0016] In an sixth aspect, the present invention provides a method for ex vivo inducing production of immune effector cells, comprising steps of: providing an activated immune cell as in any of the aforesaid first to second aspects and a lymphocyte; then co-incubating the activated immune cell and lymphocyte in a suitable medium to obtain the immune effector cell.

[0017] The objective of the present invention proves the immunogenic composition could activate anti-CMV immune response effectively, and the peptide pool has non-live virus, non-CMV-infected cell, therefore not producing potential risk such as infection, immune inhibition and immune tolerance. Moreover, the method according to the present invention could activate immune cells effectively, and further activate lymphocytes, and produce the immune cells having activity against CMV.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a schematic flow chart diagram illustrating the method of the present invention;

[0019] FIG. 2 illustrates the diagram of CMV polypeptide-derived peptide pool, with pp65 as the polypeptide;

[0020] FIG. 3 illustrates the flow chart of production of activated lymphocytes, which are activated by dendritic cells;

[0021] FIG. 4 illustrates the effect of various CMV proteins-derived peptide pools and their combinations on inducing proliferation of PBMC, wherein panel A illustrates the result of CFSE staining analysis, and panel B illustrates the result of intracellular cytokine staining analysis;

[0022] FIG. 5 illustrates the effect of various CMV proteins-derived peptide pools and their combinations on inducing immune response of PBMC, wherein panel A and B illustrate the results of various donators' PBMCs;

[0023] FIG. 6 illustrates the effect of various CMV proteins-derived peptide pools and their combination on cell proliferations of populations of CD4.sup.+ and CD8.sup.+ cells in PBMCs;

[0024] FIG. 7, in panels A and B, illustrates the cytokine secretion of PBMCs of various origins stimulated by various CMV proteins-derived peptide pools and their combinations;

[0025] FIG. 8 illustrates the result of cellular cytokine staining analysis of CMV-specific immune effector cells, which were non-adherent PBMCs stimulated by DCs pulsed with the mixed pool of 5 CMV pentadecapeptides ; and

[0026] FIG. 9 is the bar chart of the result of FIG. 8; panel A illustrates expression of IL-2 of each group, and panel B illustrates expression of CD107a of each group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The applicants of the present invention develop an immunogenic composition and a method for immune effector cells against herpes virus. With reference to FIG. 1, the method of the present invention involves the following aspects. First, using various immunogenic compositions (10) containing peptides derived from CMV peptides to stimulate immune cells (20), wherein the immunogenic composition (10), for example, includes peptide pools derived from CMV pp65, CMV IE-1, CMV VGLB, CMV VPAP and CMV p100 polypeptides (11) (12) (13) (14) (15) to obtain activated immune cells (21) with capability of activating lymphocytes such as T lymphocytes. Second, activated immune cells and lymphocytes are co-incubated to obtain immune effector cells (31). Then, the immune effector cells are further implanted into individuals (40) to achieve the purpose of preventing and treating herpes virus related diseases.

[0028] Applicants discover the mixed pools of two, three, four or five CMV peptide pools, compared with only single or double CMV protein-derived peptide pools, are more effective in activating anti-CMV immune response; especially CMV specific CD8.sup.+ and CD4.sup.+T lymphocytes are proved activated to generate high effector functions. Such reveals that CMV immunogenic composition and immune treatment of the present invention could be used to prevent and treat herpes virus related diseases.

[0029] Therefore, in one aspect, the present invention provides an immunogenic composition containing peptides derived from CMV, which comprises: (a) at least one peptide selected from CMV pp65 and CMV IE-1 polypeptides-derived peptide; and (b) one or more peptide pool(s) selected from the group consisting of peptide pools of CMV VGLB, CMV VPAP and CMVp100 polypeptides-derived peptide.

[0030] In another aspect, the present invention provides an immunogenic composition containing peptides derived from CMV, which comprises: (a) at least one peptide pool selected from CMV pp65 and CMV IE-1 polypeptides-derived peptide pools; and (b) one or more peptide pool(s) selected from the group consisting of CMV VGLB, CMV VPAP and CMVp100 polypeptides-derived peptide pools.

[0031] CMV pp65 is a CMV structural protein, which is referred to as HCMV phosphorylated matrix protein, and includes the following characteristics: (1) Name: pp65, 65 kDa lower matrix phosphoprotein, i.e. Tegument protein UL83; (2) Organism: Human cytomegalovirus, belonging to Human herpesvirus-5 (HHV-5); (3) Sequence length: 561 amino acids; and (4) Function: forming part of the matrix of the HCMV virion. In a preferred embodiment, the sequence is as set forth in serial number Swiss-Prot: P06725, NCBI Accession NO: NP.sub.--040018 or SEQ ID NO: 1.

[0032] CMV IE-1 is a CMV major immediate early protein-1, which is a transcription regulatory protein, and includes the following characteristics: (1) Name: IE-1, UL123; (2) Organism: Human cytomegalovirus, belonging to Human herpesvirus-5 (HHV-5); (3) Sequence length: 491 amino acids; and (4) Function: Immediate-early transcriptional regulator. The IE1 protein augments the activation of the E1.7 promoter by EI2. In a preferred embodiment, the sequence is as set forth in serial number Swiss-Prot: P13202 sequence, NCBI Accession NO: NP.sub.--040060 or SEQ ID NO: 2.

[0033] CMV VGLB is a CMV viral envelope glycoprotein, and includes the following characteristics: (1) Name: Glycoprotein B (gB), also named Glycoprotein GP55, and UL55; (2) Organism: Human cytomegalovirus, belonging to Human herpesvirus-5 (HHV-5); (3) Sequence length: 907 amino acids; and (4) Function: Type 1 membrane protein and also as a viral ligand for CD209/DC-SIGN to allow capturing of viral particles by dendritic cells (DCs) and subsequently transferring viruses to permissive cells. In a preferred embodiment, the sequence is as set forth in serial number NCBI Accession NO: YP.sub.--081514 or SEQ ID NO: 3.

[0034] CMV VPAP is a CMV DNA polymerase processivity factor, which is subunit of HCMV polymerase protein, and includes the following characteristics: (1) Name: DNA polymerase processivity factor, also named Polymerase accessory protein (PAP), and UL44; (2) Organism: Human cytomegalovirus, belonging to Human herpesvirus-5 (HHV-5); (3) Sequence length: 433 amino acids; and (4) Function: accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization by similarity. In a preferred embodiment, the sequence is as set forth in serial number NCBI Accession NO: AAO73452 or SEQ ID NO: 4.

[0035] CMV p100 is a CMV capsid protein, which is 150 kDa HCMV phosphoprotein and includes the following characteristics: (1) Name: pp150, also named basic phosphoprotein (BPP), and UL32; (2) Organism: Human cytomegalovirus, belonging to Human herpesvirus-5 (HHV-5); (3) Sequence length: 1046 amino acids; and (4) Function: large structural phosphoprotein. In a preferred embodiment, the sequence is as set forth in serial number NCBI Accession NO: AAO73452 or SEQ ID NO: 5.

[0036] In a preferred embodiment of the present invention, CMV pp65, IE-1, VGLB, VPAP and CMV p100 polypeptides respectively have a sequence essentially identical to their corresponding sequences as mentioned above.

[0037] According to the present invention, the term "-derived peptide fragment" refers to peptide fragment or peptide that has sequences of whole or a portion of protein.

[0038] According to the present invention, the term "essentially identical to" refers to the variation of amino acid sequence that is not necessarily affecting the activity of formed protein in various species. Therefore, said variation will not affect the activity of formed protein only if the amino acid sequence has a certain homology among identified sequences. Preferably, said certain homology is more than 70%, more preferably, having 80% of homology, and the most preferably, having 90% of homology.

[0039] In a preferred embodiment of the present invention, the above-mentioned immunogenic composition includes CMV pp65, CMV IE-1, CMV VGLB, CMV VPAP and CMV p100 polypeptide-derived peptide pools. The term "peptide pool", as used hereby, refers to a combination containing partially overlapping peptides spanning whole sequence or a portion sequence of a gene product.

[0040] In a preferred embodiment of the present invention, each peptide of the polypeptides-derived peptide pool has a length of 12 to 18 amino acids.

[0041] In a preferred embodiment of the present invention, wherein the two adjacent peptides of each peptide pool has a 10 to 15 continuous amino acid residues overlapping in sequence.

[0042] For example, if the aforesaid polypeptide-derived peptide pool includes pentadecapeptides, that means a peptide pool of 15-mer peptide spanning full-length gene product with partial amino acid overlapping in sequence, and likewise the peptide pools with peptides of other lengths.

[0043] In a preferred embodiment of the present invention, the two adjacent pentadecapeptides in the peptide pool derived from each polypeptide have 11 continuous amino acid residues overlapping in sequence. For instance, as shown in FIG. 2, the gene product of CMV pp65 gene has a sequence as set forth in SEQ ID NO: 1, including 561 amino acids. The pentadecapeptides (11) derived from CMV pp65 polypeptide include multiple peptides, wherein a 15-mer peptide (111) has a sequence of 15 continuous amino acids from the site 1.sup.st to 15.sup.th. Another adjacent 15-mer peptide (112) has a sequence of 15 continuous amino acids from the site 5.sup.th to 15.sup.th, containing 11 continuous and partially overlapping amino acids, and 4 continuous amino acid residue sequences from the site 16.sup.th to 19.sup.th. Yet another adjacent 15-mer peptide (113) has a sequence of amino acids from the site 9.sup.th to 19.sup.th, containing 11 continuous and partially overlapping amino acids and 4 continuous amino acid residues from the site 20.sup.th to 23.sup.rd, and so on. For the last sequence that has less than four continuous amino acid residues, number of its overlapping amino acids is increased to be pentadecapeptide such that each peptide of the peptide pools is 15-mer.

[0044] According to the present invention, the above principle is also adapted to the CMV IE-1, CMV VGLB, CMV VPAP and CMV p100 polypeptide-derived peptide pools.

[0045] According to the present invention, said peptide pools are prepared by methods for preparing peptide pools known in the art. In a preferred embodiment of the present invention, said peptide pools are composed of synthetic peptides.

[0046] According to the present invention, said adjacent peptides in said peptide pool refer to two peptides having the maximal overlapping continuous amino acid sequences. Take said peptide pool of 15-mer peptides for example, if the peptide pool is set to have 11 continuous amino acid sequences overlapping, the two adjacent peptides respectively have 11 continuous amino acid sequences close to C-terminal and 11 continuous amino acid sequences close to N-terminal, which are identical.

[0047] According to the present invention, said immunogenic composition further includes a physiologically acceptable carrier or excipient.

[0048] According to the present invention, said immunogenic composition further includes an immunostimulant.

[0049] The term "immunostimulant" as used hereby refers to any material, which could substantially improve or enhance external antibody- or cell-mediated immune responses against exogenous antigen. A preferable immunostimulant includes an adjuvant. The adjuvant includes materials which are designed to protect antigen from fast metabolism, such as aluminum hydroxide or mineral oil; and a stimulator of immune responses, such as copolymer of surface active agent, including lipid A. Also included are Bordetalla pertussis derived proteins and Mycobacterium tuberculosis derived proteins, and glycolipids used as Immunoregulator, such as .alpha.-galatosylceramide (.alpha.-GalCer) and derivatives, and CqG derived polynucleotides.

[0050] Specifically, adjuvants were any commercially available agent such as Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salt, such as aluminum hydroxide gel or aluminum phosphate; the salt of calcium, iron or zinc; insoluble suspension of acylated tyrosine; acylated sugars, cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and Quillaja saponaria A (Quil A); and others such as GM-CSF, Interleukin-2, -7, -12 cytokines and other similar growth factors also could be used as adjuvants.

[0051] In another aspect, the present invention provides a method for ex vivo activating immune cells, comprising steps of: mixing an immune cell and the aforesaid immunogenic composition to form a mixed cell culture; then incubating the mixed culture in a suitable medium to obtain the activated immune cell.

[0052] According to the present invention, said immune cell is an antigen-presenting cell; preferably, said immune cells include dendritic cells; preferably dendritic cells are derived from peripheral blood mononuclear cells, bone marrow cells, hematopoietic progenitor cells or stem cells.

[0053] As known by persons of ordinary skill in the art, dendritic cells are leukocytes existing in mammals, mainly in blood, exposed tissues and other tissues, such as in epithelia tissue of skin, nasal cavity, lung, stomach and intestine. The functions of dendritic cells are regulating innate and acquired immune responses induced by environmental stimulus; wherein the most important function thereof is to process antigen and then present the processed antigen to other leukocytes of immune system; therefore dendritic cells belong to antigen-presenting cells. (Science, 288: 522-527, 2000; Curr Med. Chem., 13(14): 1591-607, 2006; Nat. Rev. Cancer, 8(5): 351-60, 2008) In a preferred embodiment of the present invention, the dendritic cells are human dendritic cells.

[0054] According to the present invention, said suitable medium comprises cytokines or growth factors; the cytokines are selected from the group consisting of IL-2, IL-7 and combination thereof.

[0055] In yet another aspect, the present invention provides a method for ex vivo inducing immune effector cells, comprising steps of: providing an aforesaid activated immune cell and a lymphocyte; then co-incubating the activated immune cell and lymphocyte in a suitable medium to obtain an immune effector cell.

[0056] Preferably, the lymphocyte is an autologous or allergenic lymphocyte. More preferably, the lymphocyte is an autologous lymphocyte.

[0057] According to the present invention, the lymphocytes are derived from non-adherent peripheral blood mononuclear cells. In a preferred embodiment of the present invention, the lymphocyte is T lymphocyte or B lymphocyte; more preferably, the T lymphocyte is an autologous lymphocyte.

[0058] The term "autologous" as used hereby and known by persons of ordinary skill in the art, refers to being derived from the same individual. For instance, said autologous immune cells refer to the provided dendritic cells derived from selfsame individual, whereby avoiding unwanted immune response, such as heterologous immune response.

[0059] Based on the immunogenic composition of the present invention, which is derived from cytomegalovirus belonging to herpes virus family, the obtained activated immune cell according to the method of the present invention could also activate herpes virus-specific lymphocyte, and then be applied for inducing immune response against herpes virus.

[0060] In accordance with the present invention, the herpes virus includes, but is not limited to: cytomegalovirus, Herpes simplex virus-1 (HSV-1), Herpes simplex virus-2 (HSV-2), Varicella Zoster virus (VSV), Epstein-Barr Virus (EBV), Human herpes virus-6 (HHV-6), Human herpes virus-7 (HHV-7) and Human herpes virus-8 (HHV-8).

[0061] The present invention was further illustrated by the following examples; it should be understood that the examples and embodiments described herein are for illustrative purposes only and should not be construed as limiting the embodiments set forth herein.

Materials and Methods:

1. Peptides and Antigens

[0062] The 15-mer peptide pools were purchased from JPT Peptide Technologies GmbH (Berlin, Germany), including the mixtures of pentadecapeptides of 11 partially overlapping amino acids spanning the entire 427 amino acids of the HCMVA pp65 (SEQ ID NO:1) (561 amino acids, 138 peptides), HCMVA IE-1(SEQ ID NO:2) (491 amino acids, 120 peptides), HCMVA VPAP (SEQ ID NO:3) (433 amino acids, 106 peptides), HCMVA p100 (SEQ ID NO:4) (1048 amino acids, 259 peptides), and HCMVT VGLB (SEQ ID NO:5) (907 amino acids, 224 peptides). Other antigens and peptides can be synthesized by different methods, including WT-1 (Wilm's tumor 1)-derived peptide pool (Swiss prot: P19544; as shown by SEQ ID. NO:6) (WT33; JPT Peptide Technologies GmbH, Berlin, Germany) and core protein of Hepatitis C virus-derived peptide pool (NCBI ACCESSION: ABV46234 (1-191 //product="core protein"; as shown by SEQ ID. NO: 7), and Peptide Scan 15/11, from JPT Peptide Technologies GmbH, Berlin, Germany, all of which are used as non-specific peptide pools.

2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0063] Apheresis blood or whole blood was voluntarily donated by healthy donors. PBMCs were prepared by gradient density centrifugation in Ficoll-Hypaque (GE Healthcare Bio-Sciences AB, NJ, USA) as previously described {Han, 2008 #5292}. Viability of the human PBMCs was determined by trypan blue staining and only cells with a viability of .gtoreq.80% were used.

3. Preparation of Dendritic Cells (DCs)

[0064] PBMCs were placed into 6-well plates of 1.times.10.sup.7 cells/well and adhered for 2 hours in the medium AIM-V (Gibco-BRL, CA, USA). Then, non-adherent cells were removed gently and frozen as source of T cells for further co-culture. Adherent cells were cultured in the medium AIM-V supplemented with 50 ng/ml GM-CSF (Biosource, CA, USA) and 25 ng/ml IL-4 (Biosource, CA, USA). For the generation of DCs, cells were cultured with GM-CSF and IL-4 for 24 hours and incubated for another 24 hours with IFN-.gamma. (20 ng/ml) (Gentaur), TNF-.alpha. (50 ng/ml) (R&D systems, MN, USA), IL-1.beta. (10 ng/ml) (R&D systems, MN, USA), IL-6 (10 ng/ml) (R&D systems) and PGE2 (1 .mu.M) (Sigma-Aldrich, MO, USA) for maturation. The phenotype of DCs was analyzed with flow cytometry.

4. Dendritic Cell-Activated and Ex Vivo Expanded Antigen-Specific Immune Cells

[0065] As shown in FIG. 3, the methods were approximately processed as shown in the figure. Dendritic cell-activated immune cells were generated as previously described (Han et al, supra). In brief, mature DCs were loaded with peptides (5 .mu.g/ml) or other antigens for 3 hours and irradiated (2,500 rads). The DCs were cocultured with autologous non-adherent PBMCs at a ratio of 1:20 in AIM-V with 2% human AB serum. On Day 3, IL-2 (Gentaur, Aachen, Germany), IL-7 (Gentaur) and IL-15 (Gentaur) were added. Then, fresh medium with cytokines were added every other days. The function of T cells was analyzed by intracellular cytokine staining.

5. CFSE Cell Proliferating Analysis

[0066] Briefly, 1.times.10.sup.6 PBMCs were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester-based), and then stimulated by antigens in culture. On Day 3, IL-2 (Gentaur, Aachen, Germany) and IL-15 (Gentaur) were added. Then, fresh medium with cytokines were added every other days until Day 7 and then were subjected to flow cytometry analysis.

6. CD107a and Intracellular Cytokine Staining

[0067] The assay was performed as described (Han et al., supra). Briefly, 3.times.10.sup.5 expanded CMV immune effector cells were stimulated with antigen-loaded autologous DCs or monocytes in AIM-V with or without anti-CD107a-FITC. Monensin (Sigma) was added 1 hour after stimulation. After 5 hours, cells were stained for CD4 and CD8, fixed, permeabilized, and stained with antibodies against IFN-.gamma. (or TNF-.alpha., or IL-2) (all from BD Bioscience) using FIX/PERM and PERM/Wash solution (BD).

7. Enzyme-Linked ImmunoSpot Assay (ELISpot Assay)

[0068] Interferon-.gamma. (IFN-.gamma.) ELISPOT assays were performed by incubation of 1.times.10.sup.5 PBMCs/well with different combinations of pepmix in ninety-six-well, ELISPOT plates (Millipore, Mass.) precoated with anti-human IFN-.gamma. monoclonal antibody (1-D1K; MabTech, Sweden) and blocked by 5% inactivated human AB serum in duplicate. After 18 to 20 hours of culturing at 37.degree. C. in a humidified incubator under 5% CO.sub.2, plates were washed and stained with 1 .mu.ml biotinylated anti-human IFN-.gamma. as the secondary antibody (7-B6-1; MabTech), followed by streptavidin conjugated alkaline phosphatase (streptavidin conjugated ALP) (MabTech) and BCIP/NBT-plus substrate (Bio-Rad) to develop the color reaction. The colored spots were counted using the EliSpot Reader (AutoImmune Diagnostika). Results were presented by IFN-.gamma. spot-forming cells (SFC)/10.sup.6 PBMCs.

EXAMPLE 1

To Analyze the Effect of Peptide Pools of Various CMV Proteins and Their Combinations on Stimulating PBMC By CFSE Staining Method

[0069] The objective of the present example is to use peptide pools of various CMV proteins and their combinations to stimulate PBMC, and analyze the effect of peptide pools of various CMV-derived proteins and their combinations on proliferation of PBMC by CFSE staining, to understand the effect of various combinations on inducing immune response.

[0070] By method as described in General materials and method, CFSE-labeled PBMCs were stimulated by various peptide pools or their combinations for 7 days, as divided into the following groups: [0071] (1) The CMV pentadecapeptides of pp65 were indicated as sample 1. [0072] (2) The CMV pentadecapeptides of VGLB were indicated as sample 2. [0073] (3) The CMV pentadecapeptides of IE-1 were indicated as sample 3. [0074] (4) The CMV pentadecapeptides of p100 were indicated as sample 4. [0075] (5) The CMV pentadecapeptides of VPAP were indicated as sample 5. [0076] (6) The CMV pentadecapeptides of pp65 and VGLB were indicated as sample 6. [0077] (7) The CMV pentadecapeptides of pp65 and IE-1 were indicated as sample 7. [0078] (8) The CMV pentadecapeptides of VGLB and IE-1 were indicated as sample 8. [0079] (9) The CMV pentadecapeptides of pp65, VGLB and IE-1 were indicated as sample 9. [0080] (10) The CMV pentadecapeptides of pp65, VGLB, IE-1 and p100 were indicated as sample 10. [0081] (11) The CMV pentadecapeptides of pp65, VGLB, IE-1, p100 and VPAP were indicated as sample 11.

[0082] Further, the following groups worked as the control: [0083] (12) CFSE-labeled PBMCs were indicated as no stimulation control for background value as sample 12. [0084] (13) Co-culture of CFSE-labeled PBMCs and dendritic cells loaded with core protein of Hepatitis C virus derived peptide pool worked as the negative control, indicated as sample 13.

[0085] The above stimulated-PBMCs were subjected to antigen-specific proliferation and intracellular cytokine staining analysis by the method as described in General materials and method. The obtained results were minus value obtained from PBMCs before being plotted as a graph.

[0086] As shown in FIG. 4, panel A illustrates the situation of cell proliferation under the stimulation by various mixed peptide pools; panel B illustrates cells' abilities to express IFN-.gamma., wherein the cells proliferate under the stimulations by various mixed peptide pools.

[0087] The above results show that: (1) 5 CMV proteins-derived peptide pools individually induce responses; (2) any single polypeptide of pp65, VGLB or p100 polypeptide derived peptide pool could also induce immune response; (3) the result of two peptide pools mixed is better than only one peptide pool, and (4) the immune response induced by the peptide pool either of pp65 or VGLB is better than any of the rest CMV protein-derived peptide pool, combination of said peptide pools has additive effect on enhancing immune response, and the effect is better when more peptide pools are mixed.

EXAMPLE 2

To Analyze the Effect of Peptide Pools of Various CMV Proteins and Their Combinations on Stimulating PBMC By ELISpot Assay

[0088] The objective of the present example is to use various CMV protein-derived peptide pools and their combinations to stimulate PBMC, and analyze the effect of various CMV protein-derived peptide pools and their combinations on cellular function of PBMC by ELISpot assay.

[0089] By method as described in General materials and method, newly-isolated PBMCs were separately obtained from various donators. PBMCs were further stimulated by the following groups of various combinations of peptide, incubated for 20 hours, and processed with IFN-.gamma. analysis by ELISpot as above-mentioned in General materials and method. [0090] (1) The CMV pentadecapeptides of pp65 were indicated as sample 1. [0091] (2) The CMV pentadecapeptides of VGLB were indicated as sample 2. [0092] (3) The CMV pentadecapeptides of IE-1 were indicated as sample 3. [0093] (4) The CMV pentadecapeptides of pp65 and VGLB were indicated as sample 4. [0094] (5) The CMV pentadecapeptides of pp65 and IE-1 were indicated as sample 5. [0095] (6) The CMV pentadecapeptides of pp65, VGLB and IE-1 were indicated as sample 6. [0096] (7) The CMV pentadecapeptides of pp65 VGLB, IE-1 and p100 were indicated as sample 7. [0097] (8) The CMV pentadecapeptides of pp65, VGLB, IE-1, p100 and VPAP were indicated as sample 8.

[0098] As shown in FIG. 5, the effect of the group pp65+VGLB (as bar 4) or pp65+IE-1 (as bar 5) on stimulating PMBC to produce IFN-.gamma. is more prominent than that of pp65 (as bar 1). The stimulating effect of pp65+VGLB (bar 4) is better than pp65+IE-1 (as bar 5). And the effect of combination of various CMV protein-derived peptide pools (as bar 6, 7 or 8) is better than that of the group having only two CMV protein-derived peptide pools (as bar 4 or 5) or that of the group of pp65 alone (as bar 1).

EXAMPLE 3

To Analyze the Proliferation of Cell Populations of PBMC Stimulated By CMV Proteins-Derived Peptide Pool and Their Combinations

[0099] The objective of the present example is to use various CMV protein-derived peptide pools and their combinations to stimulate PBMC, and to analyze the effect of the CMV protein-derived peptide pools and their combinations on inducing proliferation of CD4.sup.+ and CD8.sup.+ populations in PBMC.

[0100] The CFSE-labeled PBMCs were respectively cultured with the stimulus as indicated as the following: (1) the pentadecapeptides of HCMVA pp65 was indicated as "pp65"; (2) the mixed pool of pentadecapeptides of both HCMVA pp65 and HCMVA IE-1, including HCMVA pp65 polypeptide-derived peptide pool and HCMVA IE-1 polypeptide-derived peptide pool, were indicated as "pp65/IE-1"; (3) the mixed pool of the pentadecapeptides of HCMVA pp65, HCMVA IE-1, HCMVA VPAP, HCMVA p100 and HCMVT VGLB were indicated as "5 pepmix"; (4) the nonspecific pentadecapeptides were indicated as "WT-1"; (5) anti-CD3 antibody as positive control was indicated as "anti-CD3", and, incubated for seven days, and were subjected to antigen-specific proliferation by the method as mentioned in General materials and method, especially to cell proliferation of CD4.sup.+ and CD8.sup.+ cell populations, and separately incubated CFSE-labeled PBMC as no stimulation control was indicated as "PBMC".

[0101] As shown in FIG. 6, the percentage of CFSE-diluted cells (i.e. proliferation cell) of each group was as indicated. Among the mixed pool of CMV protein-derived pentadecapeptides (pp65) or the mixed pool of two CMV-derived protein pentadecapeptides (pp65/IE-1), the mixed pool of pentadecapeptides derived from five CMV protein (5 pepmix) induced the highest proliferation of CD4.sup.+ and CD8.sup.+ groups cell populations within PBMC, demonstrating that the mixture of peptide pools derived from five CMV protein had the best immunogenicity.

EXAMPLE 4

Functional Analysis of Various Peptide Pools Derived From CMV Protein Stimulating PBMC

[0102] The present invention further analyzed the effect of CMV protein-derived peptide pools and their combinations on stimulating the cells within PBMC, and analyzed cytokinessecretion of PBMC to determine immune function and confirm the immunogenicity of various combinations of peptide pools.

[0103] The PBMCs from two donators were respectively stimulated by the stimulus as follows: (1) the pentadecapeptides of HCMVA pp65 were indicated as "pp65"; (2) the mixed pool of pentadecapeptides of both HCMVA pp65 and HCMVA IE-1, including HCMVA pp65 polypeptide-derived peptide pool and HCMVA IE-1 polypeptide-derived peptide pool, was indicated as "pp65/IE-1"; (3) the mixed pool of pentadecapeptides of HCMVA pp65, HCMVA IE-1, HCMVA VPAP, HCMVA p100 and HCMVT VGLB was indicated as "5 pepmix"; (4) the nonspecific pentadecapeptides were indicated as "WT-1"; (5) the anti-CD3 antibody as positive control was indicated as "anti-CD3", and was incubated for seven days and processed with intracellular cytokine staining (ICCS) as mentioned in General materials and method, to analyze expression of IL-2 and IFN-.gamma.. Separately incubated PBMC was used as no stimulation control and indicated as "PBMC only".

[0104] FIG. 7, panels A and B respectively expressed results of PMBCs from various origins by the above-mentioned analysis, as illustrated by the bar chart, respectively representing expression of IL-2 and/or IFN-.gamma. of PBMC. Based on the results, among the peptide pool derived from single CMV protein (pp65) or the mixed pool of peptide pool derived from two CMV proteins (pp65/IE-1), the mixed pool of peptide pools of five CMV antigen (5 pepmix) stimulated had a highest activity to stimulate PBMCs to express IL-2 and/or IFN-.gamma., demonstrating that the combination of peptide pools of five CMV proteins had the best immunogenicity.

EXAMPLE 5

To Produce CMV-Specific Immune Cells By Stimulating PMBC with Dendritic Cells (DCs)

[0105] The present example used DCs that were pulsed with the mixed pool of pentadecapeptides derived from five CMV proteins to stimulate non-adherent PBMC to evaluate its effect on generation of CMV-specific immune cells.

[0106] The dendritic cells treated by the mixed pool of pentadecapeptides derived from 5 CMV proteins and non-adherent PMBC (as the source of T lymphocytes) were prepared by the method as mentioned in General materials and method, and co-incubated (as shown in FIG. 3). After being co-incubated for 15 days, the DC-activated lymphocytes were respectively re-stimulated by the dendritic cells loaded with an individual peptide pool derived from each CMV protein (pp65, IE-1, VGLB, VPAP and p100) and their combination for five hours, meanwhile Phorbol (PMA) and Ionomycin (PMA+Ionomycin) were used to stimulate DC-activated lymphocytes as positive control and homologous dendritic cell treated with non-specific antigen was used as control. For evaluating the function of antigen specific-immune cell, the re-stimulated lymphocytes were examined by intracellular cytokine staining (ICCS) to IFN-.gamma., IL-2 and TNF-.alpha., and by the staining the cell marker of degranulation CD107a.

[0107] FIG. 8 was the result of the above intracellular cytokine staining, wherein the expression of cytokine such as IFN-.gamma., IL-2, TNF-.alpha. and CD107a was as indicated. The bar chart illustrated in panel A and panel B of FIG. 9 was plotted based on the result of FIG. 8, and respectively demonstrated the percentages of cell populations expressing IL-2 and/or TNF-.alpha., and the percentages of cell populations expressing IFN-.gamma. and/or CD107a. The result showed that activated-immune cells induced by 5 pepmix had specific response to the pentadecapeptides derived from any of CMV proteins, wherein the immune response against pp65 and VGLB was the most prominent one. The group that is re-stimulated group by dendritic cells treated with 5 pepmix induced a highest cytokine production of immune cells.

[0108] The result of the above examples demonstrated that the combined pools of pentadecapeptides derived from multiple CMV proteins (up to five proteins) could effectively activate both CD4.sup.+ and CD8.sup.+ T lymphocytes in a short period of time. The antigen-specific proliferation and the production of effector cytokine of CD4.sup.+ and CD8.sup.+ T lymphocytes were improved obviously, demonstrating that the combination of pentadecapeptides derived from CMV proteins was useful as an effective vaccine or immune treatment for establishing anti-CMV immunity. As shown in FIG. 6 and FIG. 7, the combination of pentadecapeptides derived from five CMV proteins induced proliferation and cytokine production from CD4.sup.+ and CD8.sup.+ cells in PBMC. Although pentadecapeptides derived from single or two CMV proteins could stimulate immune response, the combination of pentadecapeptides derived from five CMV proteins could generate the strongest antigen specific immune response. The function of immune effector cells was analyzed and assessed by multi-color flow cytometry staining immune effector cell cytokines and cell marker of degranulation, CD107a. As shown in FIG. 8 and FIG. 9, compared with pentadecapeptides derived from only one or two CMV proteins, the combination of pentadecapeptides derived from five CMV proteins (5 pepmix) is the most effective to induce production of immune effector cytokine and cell marker of degranulation, of CD107a, with the combination of the five CMV peptide pools being the most effective CMV vaccine or immunogen.

[0109] Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

Sequence CWU 1

1

71561PRTHuman cytomegalovirus (strain AD169)pp65(1)..(561) 1Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly1 5 10 15Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25 30Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val 35 40 45Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp 50 55 60Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr65 70 75 80Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn 85 90 95Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr 100 105 110Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val 115 120 125His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val 130 135 140Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg145 150 155 160Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys 165 170 175Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp 180 185 190Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met 195 200 205Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val 210 215 220Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu225 230 235 240Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met 245 250 255Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe 260 265 270Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 275 280 285His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu 290 295 300Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu305 310 315 320Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 325 330 335Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp 340 345 350Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr 355 360 365Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr 370 375 380Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp385 390 395 400Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys 405 410 415Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser 420 425 430Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser 435 440 445Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro 450 455 460Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala465 470 475 480Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu 485 490 495Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu 500 505 510Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu 515 520 525Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln 530 535 540Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg545 550 555 560Gly2491PRTHuman cytomegalovirus (strain AD169)IE-1(1)..(491) 2Met Glu Ser Ser Ala Lys Arg Lys Met Asp Pro Asp Asn Pro Asp Glu1 5 10 15Gly Pro Ser Ser Lys Val Pro Arg Pro Glu Thr Pro Val Thr Lys Ala 20 25 30Thr Thr Phe Leu Gln Thr Met Leu Arg Lys Glu Val Asn Ser Gln Leu 35 40 45Ser Leu Gly Asp Pro Leu Phe Pro Glu Leu Ala Glu Glu Ser Leu Lys 50 55 60Thr Phe Glu Gln Val Thr Glu Asp Cys Asn Glu Asn Pro Glu Lys Asp65 70 75 80Val Leu Ala Glu Leu Val Lys Gln Ile Lys Val Arg Val Asp Met Val 85 90 95Arg His Arg Ile Lys Glu His Met Leu Lys Lys Tyr Thr Gln Thr Glu 100 105 110Glu Lys Phe Thr Gly Ala Phe Asn Met Met Gly Gly Cys Leu Gln Asn 115 120 125Ala Leu Asp Ile Leu Asp Lys Val His Glu Pro Phe Glu Glu Met Lys 130 135 140Cys Ile Gly Leu Thr Met Gln Ser Met Tyr Glu Asn Tyr Ile Val Pro145 150 155 160Glu Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His Asp 165 170 175Val Ser Lys Gly Ala Ala Asn Lys Leu Gly Gly Ala Leu Gln Ala Lys 180 185 190Ala Arg Ala Lys Lys Asp Glu Leu Arg Arg Lys Met Met Tyr Met Cys 195 200 205Tyr Arg Asn Ile Glu Phe Phe Thr Lys Asn Ser Ala Phe Pro Lys Thr 210 215 220Thr Asn Gly Cys Ser Gln Ala Met Ala Ala Leu Gln Asn Leu Pro Gln225 230 235 240Cys Ser Pro Asp Glu Ile Met Ala Tyr Ala Gln Lys Ile Phe Lys Ile 245 250 255Leu Asp Glu Glu Arg Asp Lys Val Leu Thr His Ile Asp His Ile Phe 260 265 270Met Asp Ile Leu Thr Thr Cys Val Glu Thr Met Cys Asn Glu Tyr Lys 275 280 285Val Thr Ser Asp Ala Cys Met Met Thr Met Tyr Gly Gly Ile Ser Leu 290 295 300Leu Ser Glu Phe Cys Arg Val Leu Cys Cys Tyr Val Leu Glu Glu Thr305 310 315 320Ser Val Met Leu Ala Lys Arg Pro Leu Ile Thr Lys Pro Glu Val Ile 325 330 335Ser Val Met Lys Arg Arg Ile Glu Glu Ile Cys Met Lys Val Phe Ala 340 345 350Gln Tyr Ile Leu Gly Ala Asp Pro Leu Arg Val Cys Ser Pro Ser Val 355 360 365Asp Asp Leu Arg Ala Ile Ala Glu Glu Ser Asp Glu Glu Glu Ala Ile 370 375 380Val Ala Tyr Thr Leu Ala Thr Ala Gly Val Ser Ser Ser Asp Ser Leu385 390 395 400Val Ser Pro Pro Glu Ser Pro Val Pro Ala Thr Ile Pro Leu Ser Ser 405 410 415Val Ile Val Ala Glu Asn Ser Asp Gln Glu Glu Ser Glu Gln Ser Asp 420 425 430Glu Glu Glu Glu Glu Gly Ala Gln Glu Glu Arg Glu Asp Thr Val Ser 435 440 445Val Lys Ser Glu Pro Val Ser Glu Ile Glu Glu Val Ala Pro Glu Glu 450 455 460Glu Glu Asp Gly Ala Glu Glu Pro Thr Ala Ser Gly Gly Lys Ser Thr465 470 475 480His Pro Met Val Thr Arg Ser Lys Ala Asp Gln 485 4903907PRTHuman cytomegalovirus (strain Merlin)VGLB(1)..(907) 3Met Glu Ser Arg Ile Trp Cys Leu Val Val Cys Val Asn Leu Cys Ile1 5 10 15Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr Arg Gly Thr Ser 20 25 30Ala Thr His Ser His His Ser Ser His Thr Thr Ser Ala Ala His Ser 35 40 45Arg Ser Gly Ser Val Ser Gln Arg Val Thr Ser Ser Gln Thr Val Ser 50 55 60His Gly Val Asn Glu Thr Ile Tyr Asn Thr Thr Leu Lys Tyr Gly Asp65 70 75 80Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met 85 90 95Ala Gln Gly Thr Asp Leu Ile Arg Phe Glu Arg Asn Ile Val Cys Thr 100 105 110Ser Met Lys Pro Ile Asn Glu Asp Leu Asp Glu Gly Ile Met Val Val 115 120 125Tyr Lys Arg Asn Ile Val Ala His Thr Phe Lys Val Arg Val Tyr Gln 130 135 140Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr Ile His Thr Thr Tyr145 150 155 160Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pro Pro Met Trp Glu Ile 165 170 175His His Ile Asn Ser His Ser Gln Cys Tyr Ser Ser Tyr Ser Arg Val 180 185 190Ile Ala Gly Thr Val Phe Val Ala Tyr His Arg Asp Ser Tyr Glu Asn 195 200 205Lys Thr Met Gln Leu Met Pro Asp Asp Tyr Ser Asn Thr His Ser Thr 210 215 220Arg Tyr Val Thr Val Lys Asp Gln Trp His Ser Arg Gly Ser Thr Trp225 230 235 240Leu Tyr Arg Glu Thr Cys Asn Leu Asn Cys Met Val Thr Ile Thr Thr 245 250 255Ala Arg Ser Lys Tyr Pro Tyr His Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270Val Val Asp Ile Ser Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285Tyr Phe Gly Glu Asn Ala Asp Lys Phe Phe Ile Phe Pro Asn Tyr Thr 290 295 300Ile Val Ser Asp Phe Gly Arg Pro Asn Ser Ala Leu Glu Thr His Arg305 310 315 320Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330 335Gln Asp Glu Lys Asn Val Thr Cys Gln Leu Thr Phe Trp Glu Ala Ser 340 345 350Glu Arg Thr Ile Arg Ser Glu Ala Glu Asp Ser Tyr His Phe Ser Ser 355 360 365Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Gln Glu Val Asn Met 370 375 380Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Glu Ala Ile Asn Lys Leu385 390 395 400Gln Gln Ile Phe Asn Thr Ser Tyr Asn Gln Thr Tyr Glu Lys Tyr Gly 405 410 415Asn Val Ser Val Phe Glu Thr Thr Gly Gly Leu Val Val Phe Trp Gln 420 425 430Gly Ile Lys Gln Lys Ser Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445Ser Ser Leu Asn Leu Thr His Asn Arg Thr Lys Arg Ser Thr Asp Gly 450 455 460Asn Asn Ala Thr His Leu Ser Asn Met Glu Ser Val His Asn Leu Val465 470 475 480Tyr Ala Gln Leu Gln Phe Thr Tyr Asp Thr Leu Arg Gly Tyr Ile Asn 485 490 495Arg Ala Leu Ala Gln Ile Ala Glu Ala Trp Cys Val Asp Gln Arg Arg 500 505 510Thr Leu Glu Val Phe Lys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile 515 520 525Leu Ser Ala Ile Tyr Asn Lys Pro Ile Ala Ala Arg Phe Met Gly Asp 530 535 540Val Leu Gly Leu Ala Ser Cys Val Thr Ile Asn Gln Thr Ser Val Lys545 550 555 560Val Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser 565 570 575Arg Pro Val Val Ile Phe Asn Phe Ala Asn Ser Ser Tyr Val Gln Tyr 580 585 590Gly Gln Leu Gly Glu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr 595 600 605Glu Glu Cys Gln Leu Pro Ser Leu Lys Ile Phe Ile Ala Gly Asn Ser 610 615 620Ala Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met Ile Asp Leu Ser625 630 635 640Ser Ile Ser Thr Val Asp Ser Met Ile Ala Leu Asp Ile Asp Pro Leu 645 650 655Glu Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr Ser Gln Lys Glu Leu 660 665 670Arg Ser Ser Asn Val Phe Asp Leu Glu Glu Ile Met Arg Glu Phe Asn 675 680 685Ser Tyr Lys Gln Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro 690 695 700Leu Pro Pro Tyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly705 710 715 720Ala Ala Gly Lys Ala Val Gly Val Ala Ile Gly Ala Val Gly Gly Ala 725 730 735Val Ala Ser Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe 740 745 750Gly Ala Phe Thr Ile Ile Leu Val Ala Ile Ala Val Val Ile Ile Thr 755 760 765Tyr Leu Ile Tyr Thr Arg Gln Arg Arg Leu Cys Thr Gln Pro Leu Gln 770 775 780Asn Leu Phe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser785 790 795 800Gly Ser Thr Lys Asp Thr Ser Leu Gln Ala Pro Pro Ser Tyr Glu Glu 805 810 815Ser Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp 820 825 830Ala Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gln Ala Tyr Gln Met 835 840 845Leu Leu Ala Leu Ala Arg Leu Asp Ala Glu Gln Arg Ala Gln Gln Asn 850 855 860Gly Thr Asp Ser Leu Asp Gly Arg Thr Gly Thr Gln Asp Lys Gly Gln865 870 875 880Lys Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg 885 890 895His Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900 9054433PRTHuman cytomegalovirus (strain Towne)VPAP(1)..(433) 4Met Asp Arg Lys Thr Arg Leu Ser Glu Pro Pro Thr Leu Ala Leu Arg1 5 10 15Leu Lys Pro Tyr Lys Thr Ala Ile Gln Gln Leu Arg Ser Val Ile Arg 20 25 30Ala Leu Lys Glu Asn Thr Thr Val Thr Phe Leu Pro Thr Pro Ser Leu 35 40 45Ile Leu Gln Thr Val Arg Ser His Cys Val Ser Lys Ile Thr Phe Asn 50 55 60Ser Ser Cys Leu Tyr Ile Thr Asp Lys Ser Phe Gln Pro Lys Thr Ile65 70 75 80Asn Asn Ser Thr Pro Leu Leu Gly Asn Phe Met Tyr Leu Thr Ser Ser 85 90 95Lys Asp Leu Thr Lys Phe Tyr Val Gln Asp Ile Ser Asp Leu Ser Ala 100 105 110Lys Ile Ser Met Cys Ala Pro Asp Phe Asn Met Glu Phe Ser Ser Ala 115 120 125Cys Val His Gly Gln Asp Ile Val Arg Glu Ser Glu Asn Ser Ala Val 130 135 140His Val Asp Leu Asp Phe Gly Val Val Ala Asp Leu Leu Lys Trp Ile145 150 155 160Gly Pro His Thr Arg Val Lys Arg Asn Val Lys Lys Ala Pro Cys Pro 165 170 175Thr Gly Thr Val Gln Ile Leu Val His Ala Gly Pro Pro Ala Ile Lys 180 185 190Phe Ile Leu Thr Asn Gly Ser Glu Leu Glu Phe Thr Ala Asn Asn Arg 195 200 205Val Ser Phe His Gly Val Lys Asn Met Arg Ile Asn Val Gln Leu Lys 210 215 220Asn Phe Tyr Gln Thr Leu Leu Asn Cys Ala Val Thr Lys Leu Pro Cys225 230 235 240Thr Leu Arg Ile Val Thr Glu His Asp Thr Leu Leu Tyr Val Ala Ser 245 250 255Arg Asn Gly Leu Phe Ala Val Glu Asn Phe Leu Thr Glu Glu Pro Phe 260 265 270Arg Arg Gly Asp Pro Phe Asp Lys Asn Tyr Val Gly Asn Ser Gly Lys 275 280 285Ser Arg Gly Gly Gly Gly Gly Gly Gly Ser Leu Ser Ser Leu Ala Asn 290 295 300Ala Gly Gly Leu His Asp Asp Gly Pro Gly Leu Asp Asn Asp Leu Met305 310 315 320Asn Glu Pro Met Gly Leu Gly Gly Leu Gly Gly Gly Gly Gly Gly Gly 325 330 335Gly Lys Lys His Asp Arg Gly Gly Gly Gly Gly Ser Gly Thr Arg Lys 340 345 350Met Ser Ser Gly Gly Gly Gly Gly Asp His Asp His Gly Leu Ser Ser 355 360 365Lys Glu Lys Tyr Glu Gln His Lys Ile Thr Ser Tyr Leu Thr Ser Lys 370 375 380Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Leu Asp Arg385 390 395 400Asn Ser Gly Asn Tyr Phe Asn Asp Ala Lys Glu Glu Ser Asp Ser Glu 405 410 415Asp Ser Val Thr Phe Glu Phe Val Pro Asn Thr Lys Lys Gln Lys Cys 420 425 430Gly51046PRTHuman cytomegalovirus (strain Towne)p100(1)..(1046) 5Met Ser Leu Gln Phe Ile Gly Leu Gln Arg Arg Asp Val Val Ala Leu1 5 10 15Val Asn Phe Leu Arg His Leu Thr Gln Lys Pro Asp Val Asp Leu Glu 20 25 30Ala His Pro Lys Ile Leu Lys Lys Cys Gly Glu Lys Arg Leu His Arg 35 40

45Arg Thr Val Leu Phe Asn Glu Leu Met Leu Trp Leu Gly Tyr Tyr Arg 50 55 60Glu Leu Arg Phe His Asn Pro Asp Leu Ser Ser Val Leu Glu Glu Phe65 70 75 80Glu Val Arg Cys Ala Ala Val Ala Arg Arg Gly Tyr Thr Tyr Pro Phe 85 90 95Gly Asp Arg Gly Lys Ala Arg Asp His Leu Ala Val Leu Asp Arg Thr 100 105 110Glu Phe Asp Thr Asp Val Arg His Asp Ala Glu Ile Val Glu Arg Ala 115 120 125Leu Val Ser Ala Val Ile Leu Ala Lys Met Ser Val Arg Glu Thr Leu 130 135 140Val Thr Ala Ile Gly Gln Thr Glu Pro Ile Ala Phe Val His Leu Lys145 150 155 160Asp Thr Glu Val Gln Arg Ile Glu Glu Asn Leu Glu Gly Val Arg Arg 165 170 175Asn Met Phe Cys Val Lys Pro Leu Asp Leu Asn Leu Asp Arg His Ala 180 185 190Asn Thr Ala Leu Val Asn Ala Val Asn Lys Leu Val Tyr Thr Gly Arg 195 200 205Leu Ile Met Asn Val Arg Arg Ser Trp Glu Glu Leu Glu Arg Lys Cys 210 215 220Leu Ala Arg Ile Gln Glu Arg Cys Lys Leu Leu Val Lys Glu Leu Arg225 230 235 240Met Cys Leu Ser Phe Asp Ser Asn Tyr Cys Arg Asn Ile Leu Lys His 245 250 255Ala Val Glu Asn Gly Asp Ser Ala Asp Thr Leu Leu Glu Leu Leu Ile 260 265 270Glu Asp Phe Asp Ile Tyr Val Asp Ser Phe Pro Gln Ser Ala His Thr 275 280 285Phe Leu Gly Ala Arg Pro Pro Ser Leu Glu Phe Asp Asp Asp Ala Asn 290 295 300Leu Leu Ser Leu Gly Gly Gly Ser Ala Phe Ser Ser Val Pro Lys Lys305 310 315 320His Val Pro Thr Gln Pro Leu Asp Gly Trp Ser Trp Ile Ala Ser Pro 325 330 335Trp Lys Gly His Lys Pro Phe Arg Phe Glu Ala His Gly Ser Leu Ala 340 345 350Pro Ala Ala Asp Ala His Ala Ala Arg Ser Ala Ala Val Gly Tyr Tyr 355 360 365Asp Glu Glu Glu Lys Arg Arg Glu Arg Gln Lys Arg Val Asp Asp Glu 370 375 380Val Val Gln Arg Glu Lys Gln Gln Leu Lys Ala Trp Glu Glu Arg Gln385 390 395 400Gln Asn Leu Gln Gln Arg Gln Gln Gln Pro Pro Pro Pro Thr Arg Lys 405 410 415Pro Gly Ala Ser Arg Arg Leu Phe Gly Ser Ser Ala Asp Glu Asp Asp 420 425 430Asp Asp Asp Asp Asp Glu Lys Asn Ile Phe Thr Pro Ile Lys Lys Pro 435 440 445Gly Thr Ser Gly Lys Gly Ala Ala Ser Gly Asn Gly Val Ser Ser Ile 450 455 460Phe Ser Gly Met Leu Ser Ser Gly Ser Gln Lys Pro Thr Ser Gly Pro465 470 475 480Leu Asn Ile Pro Gln Gln Gln Gln Arg His Ala Ala Phe Ser Leu Val 485 490 495Ser Pro Gln Val Thr Lys Ala Ser Pro Gly Arg Val Arg Arg Asp Ser 500 505 510Ala Trp Asp Val Arg Pro Leu Thr Glu Thr Arg Gly Asp Leu Phe Ser 515 520 525Gly Asp Glu Asp Ser Asp Ser Ser Asp Gly Tyr Pro Pro Asn Arg Gln 530 535 540Asp Pro Arg Phe Thr Asp Thr Pro Val Asp Ile Thr Asp Thr Glu Thr545 550 555 560Ser Ala Lys Pro Pro Val Thr Thr Ala Tyr Lys Phe Glu Gln Pro Thr 565 570 575Leu Thr Phe Gly Ala Gly Val Asn Val Pro Ala Gly Ala Gly Ala Ala 580 585 590Ile Leu Thr Pro Thr Pro Val Asn Pro Ser Thr Ala Pro Ala Pro Ala 595 600 605Pro Thr Pro Thr Phe Ala Gly Thr Gln Thr Pro Val Asn Gly Asn Ser 610 615 620Pro Trp Ala Pro Thr Ala Pro Leu Pro Gly Asp Met Asn Pro Ala Asn625 630 635 640Trp Pro Arg Glu Arg Ala Trp Ala Leu Lys Asn Pro His Leu Ala Tyr 645 650 655Asn Pro Phe Arg Met Pro Thr Thr Ser Thr Thr Ser Gln Asn Asn Val 660 665 670Ser Thr Thr Pro Arg Arg Pro Ser Thr Pro Arg Ala Ala Val Thr Gln 675 680 685Thr Ala Ser Gln Asn Ala Ala Asp Glu Val Trp Ala Leu Arg Asp Gln 690 695 700Thr Ala Glu Ser Pro Val Glu Asp Ser Glu Glu Glu Asp Asp Asp Ser705 710 715 720Ser Asp Thr Gly Ser Val Val Ser Leu Gly His Thr Thr Pro Ser Ser 725 730 735Asp Tyr Asn Asp Val Ile Ser Pro Pro Ser Gln Thr Pro Glu Gln Ser 740 745 750Thr Pro Ser Arg Ile Arg Lys Ala Lys Leu Ser Ser Pro Met Thr Thr 755 760 765Thr Ser Thr Ser Gln Lys Pro Val Leu Gly Lys Arg Val Ala Thr Pro 770 775 780His Ala Ser Ala Arg Ala Gln Thr Val Thr Ser Thr Pro Val Gln Gly785 790 795 800Arg Val Glu Lys Gln Val Ser Gly Thr Pro Ser Thr Val Pro Ala Thr 805 810 815Leu Leu Gln Pro Gln Pro Ala Ser Ser Lys Thr Thr Ser Ser Arg Asn 820 825 830Val Thr Ser Gly Ala Arg Thr Ser Ser Ala Ser Ala Arg Gln Pro Ser 835 840 845Ala Ser Ala Ser Val Leu Ser Pro Thr Glu Asp Asp Val Val Ser Pro 850 855 860Val Thr Ser Pro Leu Ser Met Leu Ser Ser Ala Ser Pro Ser Pro Ala865 870 875 880Lys Ser Ala Pro Pro Ser Pro Val Lys Gly Arg Gly Ser Arg Val Gly 885 890 895Val Pro Ser Leu Lys Pro Thr Leu Gly Gly Lys Ala Val Val Gly Arg 900 905 910Pro Pro Ser Val Pro Val Ser Gly Ser Ala Pro Gly Arg Leu Ser Gly 915 920 925Thr Ser Arg Ala Ala Ser Thr Thr Pro Thr Tyr Pro Ala Val Thr Thr 930 935 940Val Tyr Pro Pro Ser Ser Thr Ala Lys Ser Ser Val Ser Asn Ala Pro945 950 955 960Pro Val Ala Ser Pro Ser Ile Leu Lys Pro Gly Ala Ser Ala Ala Leu 965 970 975Gln Ser Arg Arg Ser Thr Gly Thr Ala Ala Val Gly Ser Pro Val Lys 980 985 990Ser Thr Thr Gly Met Lys Thr Val Ala Phe Asp Leu Ser Ser Pro Gln 995 1000 1005Lys Ser Gly Thr Gly Pro Gln Pro Gly Ser Ala Gly Met Gly Gly 1010 1015 1020Ala Lys Thr Pro Ser Asp Ala Val Gln Asn Ile Leu Gln Lys Ile 1025 1030 1035Glu Lys Ile Lys Asn Thr Glu Glu 1040 10456449PRTHomo sapiensWT-1(1)..(449) 6Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro1 5 10 15Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala 20 25 30Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr 35 40 45Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro 50 55 60Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly65 70 75 80Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe 85 90 95Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe 100 105 110Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe 115 120 125Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile 130 135 140Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr145 150 155 160Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe 165 170 175Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln 180 185 190Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser 195 200 205Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp 210 215 220Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln225 230 235 240Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser 245 250 255Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu 260 265 270Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile 275 280 285His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro 290 295 300Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys305 310 315 320Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys 325 330 335Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro 340 345 350Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp 355 360 365Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln 370 375 380Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr385 390 395 400His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys 405 410 415Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val 420 425 430Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala 435 440 445Leu 7191PRTHepatitis C virusHCV_core_protein(1)..(191) 7Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala 180 185 190

Claims

1. An immunogenic composition, comprising: (a) at least one peptide pool selected from CMV pp65 and CMV IE-1 polypeptides-derived peptide pools; and (b) one or more peptide pool(s) selected from the group consisting of CMV VGLB, CMV VPAP and CMVp100 polypeptides-derived peptide pools.

2. The immunogenic composition according to claim 1, wherein the CMV pp65 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 1; the CMV IE-1 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 2; the CMV VGLB has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 3; the CMV VPAP has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 4; and the CMV p100 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 5.

3. The immunogenic composition according to claim 1, wherein the peptide pools comprise CMV pp65, CMV IE-1, CMV VGLB, CMV VPAP and CMVp100 polypeptide-derived peptide pools.

4. The immunogenic composition according to claim 1, wherein each polypeptide-derived peptide pool contains peptides having a length of 12 to 18 amino acids.

5. The immunogenic composition according to claim 1, wherein each polypeptide-derived peptide pool of the immunogenic composition includes pentadecapeptides.

6. The immunogenic composition according to claim 5, wherein the two adjacent pentadecapeptides in each polypeptide-derived peptide pool has 11 continuous amino acid residues overlapping in sequence.

7. The immunogenic composition according to claim 1, wherein two adjacent peptides of each peptide pool of the immunogenic composition has 10 to 15 continuous amino acid residues overlapping in sequence.

8. A method for activating immune cells, comprising steps of: mixing an immune cell and an immunogenic composition as claims 1 to a mixed culture; then incubating the mixed cell culture in a suitable medium to obtain the activated immune cell; wherein the immunogenic composition containing CMV-derived peptide, comprises : (a) one or more peptide pool(s) selected from CMV pp65 and CMV IE-1 polypeptides-derived peptide pools; and (b) one or more peptide pool(s) selected from the group consisting of CMV VGLB, CMV VPAP and CMVp100 polypeptides-derived peptide pools.

9. The immunogenic composition according to claim 8, wherein the CMV pp65 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 1; the CMV IE-1 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 2; the CMV VGLB has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 3; the CMV VPAP has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 4; and the CMV p100 has a sequence essentially identical to the sequence as set forth in SEQ ID NO: 5.

10. The immunogenic composition according to claim 8, wherein the peptide pools comprise CMV pp65, CMV IE-1, CMV VGLB, CMV VPAP and CMVp100 polypeptide-derived peptide pools.

11. The immunogenic composition according to claim 8, wherein each polypeptide-derived peptide pool contains peptides having a length of 12 to 18 amino acids.

12. The immunogenic composition according to claim 8, wherein each polypeptide-derived peptide pool of the immunogenic composition includes pentadecapeptides.

13. The immunogenic composition according to claim 12, wherein the two adjacent pentadecapeptides in each polypeptide-derived peptide pool has 11 continuous amino acid residues overlapping in sequence.

14. The immunogenic composition according to claim 8, wherein two adjacent peptides of each peptide pool of the immunogenic composition has 10 to 15 continuous amino acid residues overlapping in sequence.

15. The method according to claim 8, wherein the immunogenic composition further includes an immunostimulant.

16. The method according to claim 8, wherein the immune cells are derived from peripheral blood mononuclear cells, bone marrow cells, hematopoietic progenitor cells or dendritic cells of stem cells.

17. The method according to claim 8, wherein the suitable medium comprises cytokines or growth factors; the cytokines are selected from the group consisting of IL-2, IL-7 and a combination thereof.

18. A method for inducing production of immune effector cells, comprising steps of: providing an activated immune cell as claims 8 and a lymphocyte; and co-incubating the activated immune cell and the lymphocyte in a suitable medium to obtain an immune effector cell.

19. The method according to claim 18, wherein the immunogenic composition further includes an immunostimulant.

20. The method according to claim 18, wherein the immune cells are derived from peripheral blood mononuclear cells, bone marrow cells, hematopoietic progenitor cells or dendritic cells of stem cells.

21. The method according to claim 18, wherein the suitable medium comprises cytokines or growth factors; the cytokines are selected from the group consisting of IL-2, IL-7 and a combination thereof.

22. The method according to claim 18, wherein the lymphocyte is derived from non-adherent peripheral blood mononuclear cells.

23. The method according to claim 18, wherein the lymphocyte is T lymphocyte or B lymphocyte.